JP4208851B2 - Nucleic acid detection cassette and cassette injection system - Google Patents

Description

    The present invention relates to a nucleic acid detection cassette and a cassette injection system, and in particular, disposable that automatically and consistently processes from loading of a sample containing nucleic acid to nucleic acid amplification and other necessary processing and detection of a target nucleic acid. A closed detection cassette and an injection system used with the cassette.

    With the advent of nucleic acid amplification technology and the improvement of nucleic acid detection technology, various chemicals for detecting specific DNA strands have been proposed, and individual nucleic acid amplification devices and nucleic acid detection devices have been proposed. However, in nucleic acid amplification, there is a possibility of contamination by amplified nucleic acid, and since complicated operations such as heat cycle application, liquid injection, and mixing are necessary, the use of these chemicals, amplification devices, and detection devices Is limited to experimental studies.

  Conventionally, in order to solve these problems and enable nucleic acid detection in hospitals, clinical laboratories, quarantine stations, etc., processing from samples containing nucleic acids to detection of target nucleic acids is performed automatically and consistently. A disposable closed-type detection container and a detection apparatus using the same are disclosed.

  For example, Japanese Patent No. 2536945 (Patent Document 1) discloses a cuvette for amplification and detection of nucleic acids containing all necessary reagents, and Japanese Patent Application Laid-Open No. 8-62225 (Patent Document 2) has a centrifugal force. No. 9-511407 (Patent Document 3) discloses a micro flow channel and a reaction chamber formed on a solid substrate using a micro processing technique. .

  In addition, there is Japanese Patent Application No. 2003-400878 (unpublished Patent Document 4) as a related application related to the prior application related to the proposal of the present inventors. This related application discloses a nucleic acid detection cassette in which a flow path is constituted by a combination of a fixing member and a flexible member. According to this nucleic acid detection cassette, the holding flow path having a variable volume, capable of holding a reagent, and having an access path that can select an open state that can communicate with the outside of the nucleic acid detection cassette and a closed state that can be closed, and the holding flow A connected flow path that can be connected to a path and can be connected to a holding flow path, and can be selected between an open state and a closed state that can be closed; an access path opening / closing means that can maintain the access path in a closed state; There has been disclosed a nucleic acid detection cassette comprising a maintainable connection channel opening / closing means.

This nucleic acid detection cassette consists of a series of steps that combine the transfer, mixing, heating, cooling, voltage application, current measurement, etc., of the packing in the cassette, necessary from the processing of the sample containing nucleic acid to the detection of the target nucleic acid. , Can be processed automatically and consistently in all closed cassettes, and operations necessary for each process are performed from outside the cassette, and energy generation for operating power generation means, heat source, voltage generation means, etc. in the cassette It is not necessary to have a means and is suitable for disposable use.
Japanese Patent No. 2536945 JP-A-8-62225 JP 9-511407 gazette Japanese Patent Application No. 2003-400878

(Problems associated with handling various specimens)
Using various biological materials such as liquid blood, solid or viscoelastic hair roots, skin, oral mucosa, plant seeds and leaves as nucleic acid detection target specimens, There is a need for a simple and portable automatic nucleic acid detector. In addition, there is an extremely high demand for applications in which test specimens to which these biological materials are attached or which may be attached are used in directly disposable closed-type nucleic acid detection cassettes. Yes. For example, an automatic nucleic acid detection device that can automatically perform genotyping only by setting blood, oral mucosa, hair root, etc. in various sites such as criminal investigations as well as medical diagnosis and food inspection Is expected.

  However, in the nucleic acid detection cassette proposed in Japanese Patent Application No. 2003-400878 (unpublished Patent Document 4) related to the prior application, the cassette is in contrast to the holding flow path for processing various reactions and operations for nucleophilic amplification and detection. It is necessary to inject and insert the sample from the entrance / exit part of the tube, but at this time, the path from the entrance / exit part to the holding flow path is narrow and bent at right angles, so a solid specimen such as a hair root is retained. It is difficult to insert into the flow path.

  The path from the entrance / exit path portion to the holding flow path is bent at a right angle because the depth of the holding flow path is 0.degree. When the communication hole with the outside constituting the access path is formed in a flat plate-shaped fixing member. Since it is as shallow as about 5 mm, it is difficult to form a communication hole in the side wall portion of the holding channel, and it is only possible to form a communication hole in the thickness direction of the flat plate-like fixing member. The path from the entrance / exit path portion must be bent at a right angle with respect to the holding flow path formed in the above.

  By forming a communication hole that becomes the access channel on the bottom of the holding channel, it is possible to make the path from the access channel part to the holding channel straight, but the outer wall thickness of the holding channel increases, so that Heat transfer deteriorates during heating and cooling required for various reactions and operations for detection. In addition, when the packing in the holding channel is transferred and agitated, the inside of the holding channel is pressurized and depressurized. If there is a communication hole on the bottom surface of the holding channel, leakage of the packing is likely to occur. .

  As described above, in the structure of the conventional nucleic acid detection cassette, it is difficult to insert a solid specimen or a test piece to which the specimen is attached, and there is a problem that it cannot cope with various specimens.

(Problems associated with cassette miniaturization)
In a flow channel structure capable of consistently performing a series of steps from nucleic acid amplification to inspection, the following adverse effects occur when bubbles are taken into the liquid.

  In nucleic acid amplification and other heat treatments, moisture evaporates in the bubbles, resulting in fluctuations in the reagent concentration, which becomes a reaction inhibiting factor.

  If there are bubbles or gas parts, the coefficient of thermal expansion of the gas is large, leading to an increase in the pressure in the container during heating, causing liquid leakage or contamination outside the nucleic acid detection cassette.

  If there are bubbles or gas parts, the heat conductivity is small, so the heat transfer is hindered.

  When liquid is supplied by pressurization / suction, if there are bubbles, the volume of the bubbles changes, so it becomes difficult to control liquid supply.

  In order to cope with such problems, the nucleic acid detection cassette disclosed in Japanese Patent Application No. 2003-400808 (unpublished Patent Document 4) is a self-sealing port made of a known flexible member such as rubber. An example is shown in which is attached to the access path.

  When injecting the liquid, when the injection pipette is inserted into the closed hole of the self-sealing port, the injection pipette and the flexible member of the self-sealing port are always in close contact, Bubble entrainment during injection can be prevented. When the injection pipette is pulled out at the end of injection, the hole formed in the flexible member of the self-sealing port before the injection pipette completely goes out of the self-sealing port. Self-closes and prevents liquid leakage. For this reason, the nucleic acid detection cassette is practically used in combination with other access / opening / closing means.

  However, in the case of using a flexible plastic disposable pipette tip as a part directly inserted into the closed hole of the self-sealing port in the injection pipette, EPDM, silicon rubber A self-sealing port made of a flexible member such as the like needs to have a certain size. For example, when using a pipette tip having an outer diameter of about 0.5 to 1 mm, a small-sized self-sealing port having a length of 2 to 4 × width of 3 to 6 × a height of about 3 to 6 mm is used.

  This is because in a self-sealing port that is too small for the pipette tip, the pipette tip at the time of injection is inserted into the self-sealing port and the flexible member of the self-sealing port is expanded. Because the tip is also flexible, the pipette tip is bent and cannot be penetrated through the self-sealing port.

  Conversely, using a pipette tip that is too large relative to the self-sealing port, insert the pipette tip into the self-sealing port and close the hole in the flexible member of the self-sealing port. Even if the pipette tip can penetrate through the self-sealing port when it is forced to expand, the hole of the flexible member of the self-sealing port self-closes when the pipette tip is pulled out. Cannot be performed, and liquid leakage occurs.

  Therefore, when the size of the holding channel 903 is about 10 × 10 × width 0.5 mm, the self-sealing port as described above has the same size as or larger than the size of the holding channel, and the nucleic acid detection cassette It is a factor that hinders downsizing.

  Also, in nucleic acid amplification, millions of DNA copies can be synthesized, but conversely, due to its high amplification ability, there is a problem of contamination due to previously amplified products or substances entering from the outside. In other words, even a very small amount of contamination may be amplified in a large amount. Therefore, the nucleic acid detection cassette is required to be a disposable closed type. As a similar contamination countermeasure, the instruments used to inject reagents and specimens into the nucleic acid detection cassette are also required to be disposable, and at present, it is common to use inexpensive plastic disposable pipette tips Is.

  Therefore, metal needles are more rigid than plastic disposable pipette tips, so they can have a smaller outer diameter and can penetrate even relatively small self-sealing ports, but they are expensive and are not used. Has been.

  In addition, when blood is used as a specimen, there is a risk that an accident may occur in which a metal needle with blood is stabbed into the operator's human body, thereby causing an infection. For this reason, the use of metal needles is further avoided.

For the reasons described above, the self-sealing port is a bottleneck for miniaturization of the nucleic acid detection cassette. However, the conventional technology is stable with respect to the nucleic acid detection cassette instead of the self-sealing port. Thus, no means for liquid injection is disclosed.

  Furthermore, the self-sealing port needs to penetrate the self-sealing port itself while forcibly expanding the pre-closed hole of the flexible member with a thin tube such as a pipette. It is difficult to insert a solid specimen or a test piece to which the specimen is attached into the holding channel as it is.

  The present invention has been made to solve the above-mentioned problems, and its purpose is to consistently and automatically perform from the input of a sample containing nucleic acid to nucleic acid amplification and other necessary processing and detection of the target nucleic acid. It is an object of the present invention to provide a disposable closed detection cassette and an injection system for use with the cassette.

According to this invention,
In a closed type nucleic acid detection cassette having a flow channel structure capable of performing a series of steps from amplification of nucleic acid contained in a sample to inspection,
A first block formed between a fixed substrate and a flexible sheet and having a holding channel for holding a reagent for nucleic acid amplification, wherein the fixed substrate and the flexible sheet are in close contact with each other, The holding channel is defined by a close contact region surrounding the holding channel, and an insertion guide for inserting a thin tube for injecting the sample into the holding channel is formed on the fixed substrate toward the holding channel. A close contact area extending along the insertion guide to the holding flow path is formed so as to be peelable, and an entrance / exit path is formed between the fixed substrate and the flexible sheet as the thin tube is inserted into the insertion guide. A first block ;
A retraction channel formed between the fixed substrate and the flexible sheet is provided, and the retraction channel is connected to the holding channel via a channel formed between the fixed substrate and the flexible sheet. A second block, and
A nucleic acid detection chip for nucleic acid detection fixed with a seal provided with a detection channel in the second block;
A contact plate portion provided on the flexible sheet on both sides of the insertion guide, and for tightly attaching the thin tube inserted into the insertion guide to the flexible member;
Provided on the flexible sheet between the insertion guide and the holding flow path, allowing the formation of the access path with insertion of the capillary into the insertion guide, and from the insertion guide of the capillary A closing plate portion that closes the entrance / exit path by bringing the flexible sheet into close contact with the fixed substrate as it is pulled out;
A nucleic acid detection cassette characterized by comprising:

According to this invention,
In a closed type nucleic acid detection cassette having a flow channel structure capable of performing a series of steps from amplification of nucleic acid contained in a sample to inspection,
A first block formed between a fixed substrate and a flexible sheet and having a holding channel for holding a reagent for nucleic acid amplification , wherein the fixed substrate and the flexible sheet are in close contact with each other, The holding channel is defined by a close contact region surrounding the holding channel, and an insertion guide for inserting a thin tube for injecting the sample into the holding channel is formed on the fixed substrate toward the holding channel. A close contact area extending along the insertion guide to the holding flow path is formed so as to be peelable, and an entrance / exit path is formed between the fixed substrate and the flexible sheet as the thin tube is inserted into the insertion guide. A first block ;
A retraction channel formed between the fixed substrate and the flexible sheet is provided, and the retraction channel is connected to the holding channel via a channel formed between the fixed substrate and the flexible sheet. A second block, and
A nucleic acid detection chip for nucleic acid detection fixed with a seal provided with a detection channel in the second block;
A contact plate portion provided on the flexible sheet on both sides of the insertion guide, and for tightly attaching the thin tube inserted into the insertion guide to the flexible member;
Provided on the flexible sheet between the insertion guide and the holding flow path, allowing the formation of the access path with insertion of the capillary into the insertion guide, and from the insertion guide of the capillary A closing plate portion that closes the entrance / exit path by bringing the flexible sheet into close contact with the fixed substrate as it is pulled out;
Nucleic acid detection cassette having a are used by being mounted,
A pressing means for pressing or releasing the contact plate portion, allowing the capillary tube to be inserted into the insertion guide by releasing the contact plate portion, and pressing the contact plate portion to move the capillary tube to the flexible member. Pressing means for tightly contacting
A closure pressing means to the closure plate portion in the open state or the closed state, to allow the formation of the and out passage in an open state of the closing plate portion, the flexible closed state of the closing plate portion A closing pressing means that closes the access path by bringing a sheet into close contact with the fixed substrate ;
A cassette injection system is provided comprising:

  Furthermore, according to the present invention, there is a capillary used for the above-described nucleic acid detection cassette sample injection system, the capillary having a flat outer portion, and the sample is placed in the holding channel by the capillary. When injecting, the flat part is inserted into the access path of the cassette, and the flexible member is brought into close contact with the periphery of the flat part of the thin tube by the pressing means for close contact with the thin tube. A capillary tube for use in a nucleic acid detection cassette sample injection system is provided.

  According to the present invention, in a nucleic acid detection cassette and a nucleic acid detection cassette injection system capable of automatically and consistently processing nucleic acid amplification and other necessary processing and detection of a target nucleic acid, not only liquid but also solid or The viscoelastic body can be injected into the nucleic acid detection cassette, and the nucleic acid detection cassette can be miniaturized.

  Hereinafter, a nucleic acid detection cassette and a cassette injection system according to an embodiment of the present invention will be described with reference to the drawings as necessary.

(First embodiment)
(Whole cassette)
<Basic structure>
The nucleic acid detection cassette and the cassette injection system according to the embodiment of the present invention are basically the features of the prior application except for the entrance / exit portion for injecting or discharging reagents, specimens and the like to the nucleic acid detection cassette and the injection / discharge process. The same configuration and operation method as the nucleic acid detection cassette disclosed in Japanese Patent Application No. 2003-400878 are used.

  FIG. 1 is a schematic perspective view showing a nucleic acid detection cassette 100 according to the first embodiment of the present invention. FIG. 2 is a perspective view showing the components of the nucleic acid detection cassette 100 shown in FIG. 1 separately. The nucleic acid detection cassette 100 is roughly a flow channel closed type cassette including a fixed substrate 1, a flexible sheet 2, and a cover plate 3. The fixed substrate 1 is composed of a fixed member and forms a continuous flow path. The flexible sheet 2 covers the upper surface of the flow path formed by the fixed substrate 1 and is composed of a flexible member. The cover plate 3 includes a pressing module 4 that supports the flexible sheet 2 while sandwiching it between the fixed substrate 1 and presses and deforms the flexible sheet 2 locally.

  The fixed substrate 1 is made of a polymer material such as polypropylene, polycarbonate, POM, or PMMA, silicon, glass, ceramics, or a metal such as stainless steel or aluminum. The flexible sheet 2 is made of a polymer elastomer such as silicon rubber, polypropylene rubber, or urethane rubber. The cover plate 3 is made of a polymer material such as polypropylene, polycarbonate, POM, or PMMA, or a metal such as silicon, stainless steel, or aluminum. When the fixed substrate 1, the flexible sheet 2, and the cover plate 3 are composed of a plurality of parts, different materials may be selected. The pressing module 4 is made of a polymer material such as polypropylene, polycarbonate, POM, or PMMA, silicon, glass, ceramics, or a metal such as stainless steel or aluminum.

<Block configuration>
The nucleic acid detection cassette 100 is blocked by its function. 2 has an end block 102, two intermediate blocks 101, a detection unit block 106, an intermediate block 101, and an end block 103 in order from the upstream side, that is, from the left side of FIG. It is composed of The detection block 106 is a block for nucleic acid detection, and the other end blocks 102 and 103 and the intermediate block 101 are blocks for various reactions other than nucleic acid detection. These blocks are integrally joined to each other so that a flow path provided on the surface thereof is connected. Each block may be connected by a fastening member or a fastening portion (not shown) so as to be connected and joined, or may be integrally formed by integrating the blocks.

  Practically, the fixed substrate 1, the flexible sheet 2, and the cover plate 3 are integrally formed using a polymer material. Further, when the cover plate 3 is made of a metal material, it can be processed from a single plate-like member.

<Joint>
A groove is provided on the upper surface of the fixed substrate 1. The flexible sheet 2 is disposed so as to cover the groove, and the lower surface of the flexible sheet 2 is brought into close contact with the upper surface of the fixed substrate 1 other than the groove, and the groove is closed by adhering or welding. Function as. That is, the bottom and side surfaces of the groove are constituted by the fixed substrate 1, and the upper part of the groove is covered with the flexible sheet 2 and formed in the flow path. The grooves provided on the end blocks 102 and 103 and the intermediate block 101 function as the holding channel 111, and the grooves provided on the detection unit block 106 function as the retreat channel 131.

  The holding channel 111 holds a reagent or sample used for nucleic acid amplification and detection. The retreat channel 131 is used as a temporary retreat place for the gas or liquid in the detection channel 521.

  The holding channels 111 and the retracting channels 131 are connected to each other by a connecting channel 117. The lower surface of the cover plate 3 is adhered to, adhered to, or welded to the upper surface of the flexible sheet 2. Thereby, by the pressing of the pressing module 4 provided on the upper surface of the cover plate 3, the flexible sheet 2 of the corresponding pressing portion can be bent and the flow path can be closed.

  The pressing module 4 is fastened to the cover plate 3 with fastening parts such as screws and pins, is assembled in a fitting manner, or is bonded and welded. When the pressing module 4 and the cover plate 3 are both formed of a polymer material, the pressing module 4 and the cover plate 3 can be integrally formed.

<Flow path main dimensions>
The dimension example of each structure shown by the said FIG.2 and FIG.3 is as follows.

  The holding channel 111 and the retracting channel 131 have a depth of about 0.5 mm, a length in a direction toward the adjacent channel, that is, a length between the channel start end and the channel end. The length in the direction perpendicular to the direction toward the adjacent flow path is 10 mm, that is, the flow path width is approximately 10 mm, and the standard holding volume is approximately 48 μl. When the pressing block 4 opens to the outside, the flexible sheet 2 can expand outward, for example, to maintain a holding volume that is about 2-3 times the standard holding volume. The volume of the retreat channel 131 is maintained in a reduced state before the start of the nucleic acid detection operation, and can be expanded at the start of the detection operation. The difference between the channel volume at the time of expansion and the reduction is detected. It is set to be equal to or greater than the volume of the packing in the flow path.

  The dimension of the connection channel 117 is about 0.25 mm in depth, about 2 to 6 mm in the direction toward the adjacent channel, and the length in the direction perpendicular to the direction toward the holding channel, that is, about the channel width. A fully closed state in which the flow path volume is almost zero at 2 mm can be set.

  Further, when the flexible sheet 2 is bent, an excessive internal stress or the like is not generated, and the flexible sheet 2 is set to be in close contact with the bottom surface of the flow path so that the flow volume is almost zero. Each of the cross sections is composed of a smooth curve from the bottom to the side.

  The thickness of the flexible sheet 2 is 0.2 to 0.5 mm, and both the relatively high hardness of rubber hardness JIS-A 20 ° to 30 ° and the soft one of Asker C 20 ° to 40 ° hardness are low. It can be used.

(Detection unit)
<Block configuration>
The nucleic acid detection chip 500 is fixed to the lower surface of the detection unit block 106 with a detection flow path seal 520 provided with a detection flow path interposed therebetween. A detection channel 521 is formed in a space sandwiched between the detection unit block 106, the detection channel seal 520, and the nucleic acid detection chip 500. The detection unit block 106 is provided with two contact portion openings 151 formed so as to penetrate from the front surface to the back surface. By inserting an electrical connector into the contact opening 151 and contacting the exposed contact surface of the nucleic acid detection chip 500, an electrical signal can be extracted from the chip surface.

<Nucleic acid detection method>
As long as the target nucleic acid detection method uses a single-stranded nucleic acid probe having a base sequence complementary to the target nucleic acid to be detected, immobilized in the detection channel, a known optical method, An electrochemical method or the like can be used.

  In this embodiment, basically, the electrochemical detection method disclosed in Registration No. 2573443 is applied. When using an optical system, it can respond by producing the nucleic acid detection cassette 100 using a light-transmitting material.

<Nucleic acid detection chip>
The nucleic acid detection chip 500 that is a detection sensor uses a substrate made of a fixing member. More specifically, a nucleic acid detection sensor as disclosed in JP 2002-195997 A is used. Although the detection method, the material used, the electrode structure, and the like are the same, only the structure of the detection substrate adopts the structure shown as the conventional example of the publication.

  The nucleic acid detection chip 500 has a structure in which a nucleic acid-immobilized electrode, a counter electrode, and a reference electrode are formed on a glass substrate as disclosed in JP-A-2002-195997. Further, on the glass substrate, as contacts for inputting and outputting electrical signals between these electrodes and the inspection apparatus, a contact for nucleic acid immobilization electrode, a contact for counter electrode, and a contact for reference electrode are formed, respectively.

  Further, a single-stranded nucleic acid probe having a base sequence complementary to the target nucleic acid to be detected is immobilized on the nucleic acid-immobilized electrode by the method disclosed in JP-A-2002-195997. Yes.

(Entry / exit path: function separation type pressing plate)
<Constitution>
FIG. 3 is a perspective view showing the components of the intermediate block 101 of the closed cassette 100 of the present invention separately. With reference to FIG. 1, the structure of the entrance / exit path for reagent and specimen injection will be described. However, it should be noted that the pressing module 4 corresponding to the intermediate block 101 is omitted in FIG. Further, as will be described below, the entrance / exit path portion is configured by using the end block 102 and the end block 103, and the end block 102 and the end portion are similarly applied to all of the following modified examples and embodiments. Formed as part of block 103. In FIG. 3, the flexible sheet 2 does not show the whole, only the part necessary for the description corresponding to the intermediate block 101 is shown, and similarly, the cover plate 3 does not show the whole and shows the middle. Note that only the parts required for the description corresponding to block 101 are shown.

  As shown in FIG. 3, an insertion guide 121 for a reagent and specimen injection capillary 601 is formed on the channel substrate 1 as a fixing member, in addition to the holding channel 111 and the connection channel 117. .

  The cover plate 3 is formed with a holding channel opening 311, an entrance / exit opening 312, and a connecting channel opening 317. The holding channel opening 311 and the entrance / exit opening 312 are connected by an introduction channel opening 313 to form one opening portion.

  The cover plate 3 is in close contact with the flow path substrate 1 by fastening means (not shown) except for the portions facing the openings and the connection flow path. A flow path filling such as gas, solid, or viscoelastic body does not leak out of the closed cassette 100.

  As shown in FIGS. 2 and 3, the pressing module 4 corresponding to the intermediate block 101 is attached to the upper surface of the intermediate plate 301.

<Pressing plate>
The entrance / exit path pressing means also functions as an entrance / exit path opening / closing means, and includes a closing plate 321 serving as an entrance / exit path closing pressing means and a contact plate 322 divided into two parts serving as narrow tube contact pressing means. It consists of a separate plate. Although the closing plate 321 and the contact plate 322 are separate from the cover plate 3, the surface in contact with the flexible sheet 2 is disposed on the same plane.

  The closing plate 321 and the contact plate 322 are made of a polymer material such as polyacetal, nylon, polypropylene, polycarbonate, POM, or PMMA, silicon, glass, ceramics, or a metal such as stainless steel or aluminum.

  The closing plate 321 and the close contact plate 322 are fastened to the cover plate 3 or the flow path substrate 1 so as to be movable by fastening parts such as screws and pins, or are assembled in a fitting manner.

  When the closing plate 321, the contact plate 322, and the cover plate 3 are formed of a plastic material, the closing plate 321, the contact plate 322, and the cover plate 3 can be integrally formed.

  In the structure shown in FIG. 3, the closing plate 321 and the contact plate 322 are movably attached to the flow path substrate 1 by pressing screws (fastening screws) 331 and 332. By tightening the pressing screws 331 and 332, the closing plate 321 and the contact plate 322 can individually press a predetermined amount of the area portion of the flexible sheet 2 at the lower portion thereof against the flow path substrate 1. By loosening the pressing screws 331 and 332, the pressing of the closing plate 321 and the contact plate 322 pressing the area portions of the flexible sheet 2 at the lower part against the flow path substrate 1 can be released by a certain amount. it can. In order to fasten the press screws 331 and 332, for example, holes are formed in the flexible sheet 2 corresponding to the press screws 331 and 332, and the flow path substrate 1 is provided with the press screws 331 and 332. A female screw portion to be screwed is formed. Note that these holes and internal threads are not shown in FIG.

<Injection tubule>
As the injection thin tube 601, a well-known flexible plastic disposable pipette tip is used. For example, for the flexible sheet 2 made of silicon rubber having a thickness of about 0.5 to 1 mm, an injection capillary 601 having an outer diameter of about 0.5 to 1 mm can be used. In FIG. 1, only the pipette tip is shown as the injection capillary 601, and the pipette body is omitted.

  The injection capillary 601 includes a straight needle-like insertion portion 601A that can be inserted between the fixed substrate 1 and the flexible sheet 2 and a base portion 601B having a larger diameter than the insertion portion 601A. In this injection capillary 601, since a step (step) is formed between the insertion portion 601 </ b> A and the base portion 601 </ b> B, the base portion 601 </ b> B is inserted when inserted between the fixed substrate 1 and the flexible sheet 2. Is brought into contact with the wall surface of the insertion guide 12 and only the insertion portion 601 </ b> A is inserted between the fixed substrate 1 and the flexible sheet 2. Accordingly, it is possible to prevent a situation in which only the predetermined length of the injection thin tube 601 is always inserted and the injection thin tube 601 is unnecessarily inserted to damage the flexible sheet 2.

(Liquid injection)
<Liquid injection process>
Referring to FIGS. 3, 4 (a-1) to (c-2) and FIGS. 5 (d-1) to (f-2), a liquid 602 such as a specimen or a reagent is used by using the injection thin tube 601. A process of injecting into the holding channel 111 will be described. 4 (a-1), (b-1), (c-1) and FIGS. 5 (d-1), (e-1), and (f-1) are shown in the direction in which the injection capillary 601 is inserted. The cross section of the intermediate | middle part block 101 along is shown. Here, since the injection thin tube 601 is inserted along the centers of the insertion guide 121 and the holding channel 111, these drawings are intermediate blocks along the center of the insertion guide 121 and the holding channel 111. 101 is also a cross-sectional view of FIG. 4 (a-2), (b-2), and FIG. 5 (f-2) show the approximate center of the contact plate 322 orthogonal to the direction in which the injection capillary 601 is inserted as viewed from the insertion guide 121 side. FIG. 4 (c-2), FIG. 5 (d-2), and FIG. 5 (e-2) show a cross-sectional view of the intermediate block 101 in the vicinity, and the injection capillary 601 viewed from the insertion guide 121 side is inserted. A cross-sectional view of the intermediate block 101 in the vicinity of the approximate center of the closing plate 321 perpendicular to the direction, that is, the introduction passage opening 313 is shown.

  The injection tubule 601 is inserted from the insertion guide 121 through the following process, inserted into the entrance / exit between the fixed substrate 1 and the flexible sheet 2, and then taken out.

  First, as shown in FIGS. 4A-1 and 4A-2, the pressing screws 332 of the two contact plates 322 are loosened by a predetermined amount, and the injection capillary 601 flows along the insertion guide 121. An access path is formed by being inserted between the road substrate 1 and the flexible sheet 2. At this time, since the closing plate 321 is pressed against the flexible sheet 2, the injection capillary 601 can enter only to a position before the closing plate 321.

  The holding channel 111 is in an injection standby state when the flexible sheet 2 is pressed against the channel substrate 1.

Sealing around injection capillaries:
As shown in FIGS. 4 (b-1) and 4 (b-2), the pressing screw (fastening screw) 332 is tightened by a predetermined amount so that the two contact plates 322 are formed between the flexible sheet 2 and the injection thin tube 601. It is pressed to close the gap. By the pressing of the contact plate 322, it is possible to prevent gas entrainment and liquid leakage at the time of injection and at the time of pulling out the injection thin tube.

Introductory route opening:
As shown in FIGS. 4 (c-1) and (c-2), when the pressing screw 331 of the closing plate 321 is loosened by a predetermined amount, it is pressed against the closing plate 321 by the outer diameter portion of the injection capillary 601. The partial area of the flexible sheet 2 is lifted, and the flexible sheet 2 is separated from the flow path substrate 1 to open the injection introduction path 314 which is also an entrance / exit path. Here, since the introduction path 314 is restrained by both sides of the cover plate 3 that defines the introduction path opening 313, the introduction path 314 is restricted to a predetermined size. For example, when the flexible sheet 2 is made of silicon rubber having a thickness of 0.5 to 1 mm and a rubber hardness of JIS-A 30 °, and the injection thin tube 601 is made of polypropylene having an outer diameter of 0.3 to 0.5 mm. The height of the gap between the flexible sheet 2 and the flow path substrate 1 corresponding to the gap of the introduction path 314 can be approximately 0 to 0.1 mm at the end point of the introduction path 314 that contacts the holding flow path 111. .

Injection:
As shown in FIGS. 4 (d-1) and (d-2), the liquid 602 is injected into the holding channel 111 from the injection thin tube 601 through the introduction path 314. At this time, the gap height between the flexible sheet 2 and the flow path substrate 1 is maintained substantially at the end point of the introduction path 314 that contacts the holding flow path 111 immediately before the liquid 602 is injected. . When the liquid 602 is injected, the flexible sheet 2 is separated from the flow path substrate 1 at the end point of the introduction path 314 by the pressure of the liquid 602 to secure the introduction path 314. Here, before the start of injection, the pressing module 4 is opened, and the flexible sheet 2 arranged in the holding channel 111 is separated from the channel substrate 1 by the liquid that has entered the holding channel 111. The pressure on the sheet 2 is partially released.

  Various conditions such as the size and shape of the closing plate 321, the contact plate 322, the introduction passage opening 313, and the pressing force by the pressing screws 331 and 332 with respect to the dimensions, material, hardness, etc. of the flexible sheet 2 and the injection capillary 601. Is optimized.

  Further, by making the gap between the flexible sheet 2 and the flow path substrate 1 near the tip of the injection capillary 601 as small as possible, small bubbles generated during liquid injection are captured in the vicinity of the injection capillary 601. Thus, bubbles can be prevented from entering the holding channel 111.

Introductory route closure:
As shown in FIGS. 4 (e-1) and 4 (e-2), when a predetermined amount of liquid 602 has entered, the pressing screw 331 of the closing plate 321 is tightened by a predetermined amount, and the closing plate 321 is one of the flexible sheets 2. By being pressed against the partial area, the flexible sheet 2 and the flow path substrate 1 are brought into close contact with each other, the introduction path 314 is closed, and the holding flow path 111 is sealed. At this time, a small amount of residual liquid 604 may remain in the introduction path 314, but this can be sucked by the injection thin tube 601.

Pulling out capillary tube for injection:
As shown in FIGS. 4 (f-1) and (f-2), the pressing screws 332 of the two contact plates 322 are loosened by a predetermined amount, and the injection capillary 601 is formed between the flow path substrate 1 and the flexible sheet 2. It is pulled out from between. At this time, since the introduction channel 314 is already closed and the holding channel 111 is sealed, bubbles do not enter the holding channel 111 by the drawing operation of the injection capillary 601. Thereafter, preferably, the pressing screws 332 of the two contact plates 322 are tightened by a predetermined amount, and the contact between the flow path substrate 1 and the flexible sheet 2 is reliably ensured.

  By using the liquid injection process as described above, bubbles can be prevented from entering the holding channel 111 at the time of liquid injection and at the time of pulling out the injection thin tube 601.

<Discharge process>
4 and 5 for each step of the process of discharging the liquid or gas such as the specimen or reagent already filled in the holding channel 111 from the closed cassette 100 using the injection thin tube 601. explain.

Insert tubule for injection:
Insertion of the injection thin tube 601 is the same as the injection step shown in FIGS. 4 (a-1) and (a-2).

Sealing around injection tube:
Sealing around the injection capillary 601 is the same as the injection process shown in FIGS. 4B-1 and 4B-2.

Introductory route opening:
The opening of the introduction path 314 is the same as the injection process shown in FIGS. 4 (c-1) and (c-2).

Emission:
As shown in FIG. 6A, the liquid in the holding channel 111 is sucked using the injection thin tube 601. At this time, the liquid or gas filled in the holding channel 111 is liquid or gas by the pressing module 4 pressing the flexible sheet 2 against the channel substrate 1 to reduce the volume of the holding channel 111. Is forcibly discharged.

Introductory road closure:
The closing of the introduction path 314 is the same as the injection step shown in FIGS. 5 (e-1) and (e-2).

Pulling out capillary tube for injection:
The drawing of the injection thin tube 601 is the same as the injection step shown in FIGS. 5 (e-1) and (e-2).

  Therefore, even in the discharge process, the gap between the flexible sheet 2 and the flow path substrate 1 in the vicinity of the injection thin tube 601 is extremely small, and it is possible to prevent a new gas from entering the flow path 111. Can do.

  The discharging process is also applied to the following cases.

Setting of injection standby state:
When the closed cassette 100 is assembled, the gas in the holding channel 111 is initially kept in the gas. Therefore, using the discharge process, the pressing module 4 presses the flexible sheet 2 against the flow path substrate 1 to discharge all the initial gas in the holding flow path 111. In a state where the introduction path 314 is closed and the holding flow path 111 is closed after all the gas is discharged, the flexible sheet 2 is pressed against the flow path substrate 1 by the atmospheric pressure even if the pressing of the pressing module 4 is released. It is left. This is used to set the injection standby state.

Product collection:
When a plurality of processes such as nucleic acid extraction, amplification, and detection from a specimen are consistently and automatically processed with one closed cassette 100, the products in each process are inspected, and each process is properly performed. It may be determined whether it has been done. For example, a sample is taken from the closed cassette 100 to check whether or not the target nucleic acid is synthesized to a target concentration in the nucleic acid amplification process. In this case, if a trace amount of product sample is collected from the holding channel 111 to the extent that it does not affect the next process, a consistent automatic detection process step is not hindered. At this time, a discharge process is used so that a new gas does not enter the holding channel 111. Such sample collection is used, for example, in the case of optimizing the composition of various reagents and the temperature conditions of the reaction, or for sampling and inspection of the closed-type cassette 100 in which all the reagents have been prioritized.

  In addition, when the closed-type cassette 100 is used to perform a plurality of necessary nucleic acid extraction and amplification processes and the product is used outside the closed-type cassette 100 for other purposes, the entire amount of product is collected. Will be. In this case, the closed cassette 100 is used as a general-purpose reaction container, not as a closed cassette for an automatic nucleic acid detection apparatus. Such total sampling is used, for example, in order to optimize the mixing and reaction temperature conditions of various reagents with respect to the shape and material of the closed cassette 100.

Forced discharge of bubbles:
When injecting a liquid such as a specimen or a reagent into the holding channel 111 in the injection step, the air bubbles do not enter the normal holding channel 111 by using the closing plate 321 and the contact plate 322 described above. However, if the gas dissolved in the already injected liquid appears as bubbles due to reduced pressure, overheating, etc., or if bubbles already exist in the liquid to be injected, Bubbles appear in the path 111. In such a case, as shown in FIG. 6B, the closed cassette 100 is arranged upright with the insertion guide 121 facing upward, so that the bubbles 603 in the holding channel 111 are introduced. The liquid portion 602 containing bubbles and bubbles collected by the channel 314 and sucked by the injection capillary 601 can be forcibly discharged.

<Sample injection>
The main method of using the closed cassette 100 is to simply inject the sample into the flow path 111 and then automatically detect the nucleic acid. In this case, as shown in FIG. 7A, a liquid sample 615 such as blood is injected in advance into a holding channel 111 in which all reagents 614 such as nucleic acid extraction and amplification are filled at the time of shipment. Even at the time of such sample injection, the amount of the sample to be newly injected is, for example, about 1 μl of the sample blood with respect to 47 μl of the reagent. Therefore, the liquid injection process can be used, and the flexible sheet 2 in the vicinity of the injection thin tube 601 is used. And the flow path substrate 1 are extremely small, so that it is possible to prevent a new gas from entering the flow path 111 or leakage of the liquid that is already held in the flow path 111.

  Further, when silicon rubber having a rubber hardness of about JIS-A 20 ° to 30 ° is used as the flexible sheet 2, as shown in FIG. 7B, the standard holding volume of the flow path 111 exceeds about 48 μl. Since the flexible sheet 2 can swell outside the closed cassette 100, even if the total amount of the sample 615 to be injected and the filled reagent 614 exceeds about 48 μl of the standard holding volume and reaches about 60 μl, A liquid injection process can be used. In this case, in the entrance / exit path blocking mechanism of the closed cassette 100 using the closing plate 321 and the close contact plate 322 disclosed in the present invention, by increasing the pressing pressure of the close plate 321 and the close contact plate 322, It is possible to prevent leakage of the liquid specimen and the reagent held in the specimen 111 when the specimen is injected.

<Easy to downsize>
As described above, the closing plate 321 and the two-part contact plate 322 are pressed so as to close the gap between the flexible sheet 2 and the flow path substrate 1 near and around the tip of the injection capillary 601. It is possible to prevent bubbles from entering the holding channel 111 and leaking the liquid at the time of liquid injection / discharge and at the time of the pull-out operation of the injection thin tube 601. This eliminates the need for a self-sealing port that has been used to inject liquid into the closed cassette 100 in the past.

  In addition, the size of the planar surfaces of the closing plate 321 and the contact plate 322 can be made smaller than the scale substantially the same as that of the holding channel 111, and the thickness thereof can be made smaller than the scale almost the same as that of the cover plate 3. It is about 1 to 0.5 mm. Therefore, by using the closing plate 321 and the contact plate 322 divided into two parts, the size of the closing cassette 100 can be easily reduced.

(Solid injection)
<Solid tube for solid injection>
For injection of a solid or viscoelastic body into the holding channel 111, a solid injection capillary 610 as shown in FIGS. 8 and 9 is used. The solid injection thin tube 610 includes a cylinder 611 having an outer diameter of 0.5 mm and an inner diameter of approximately 0.3 mm and a piston 612 having an outer diameter of approximately 0.3 mm, and holds a solid or viscoelastic specimen in the cylinder 611. The solid injection thin tube 610 is made of a metal material such as stainless steel or a plastic material such as polypropylene, nylon, or polyacetal. Since there is no pressure propagation to the tip located away from the piston like liquid, the solid or viscoelastic specimen is first held in the cylinder 611 and pushed out by the piston 612 at the time of injection and directly held. It is thrown into the path 111.

<Solid injection process>
There is a possibility that specimens such as hair roots, skin, oral mucosa, plant seeds and leaves, or biological materials thereof, which are solid or viscoelastic, are attached to the holding channel 111 into which a predetermined amount of reagent has been injected in advance. Each step of the process of injecting a certain test piece will be described with reference to FIGS. 8 (a-1), (b-1), (c-1) and FIGS. 9 (d-1), (e-1), and (f-1) are shown in the direction in which the injection capillary 610 is inserted. The cross section of the intermediate | middle part block 101 along is shown. Here, since the injection narrow tube 610 is inserted along the centers of the insertion guide 121 and the holding channel 111, these drawings are intermediate blocks along the center of the insertion guide 121 and the holding channel 111. 101 is also a cross-sectional view of FIG. 8 (a-2), (b-2), and FIG. 9 (f-2) show the approximate center of the contact plate 322 orthogonal to the direction in which the injection thin tube 610 is inserted as viewed from the insertion guide 121 side. FIG. 8 (c-2), FIG. 9 (d-2), and FIG. 8 (e-2) show a cross-sectional view of the intermediate block 101 in the vicinity, and the injection thin tube 610 viewed from the insertion guide 121 side is inserted. A cross-sectional view of the intermediate block 101 in the vicinity of the approximate center of the closing plate 321 perpendicular to the direction, that is, the introduction passage opening 313 is shown.

Capillary insertion for solid injection:
As shown in FIGS. 8 (a-1) and 8 (b-1), the pressing screws 332 of the two contact plates 322 are loosened by a predetermined amount, and the injection capillary 610 is moved along the insertion guide 121 into the solid injection capillary. 610 is inserted between the flow path substrate 1 and the flexible sheet 2. At this time, since the closing plate 321 is pressed, the solid injection thin tube 610 can only enter the position before the closing plate 321. At this time, a predetermined amount of liquid reagent 614 is injected into the holding channel 111 in advance.

Sealing around thin tubes for solid injection:
As shown in FIGS. 8B-1 and 8B-2, the two contact plates 322 are configured so that the pressing screw 332 is tightened by a predetermined amount to close the gap between the flexible sheet 2 and the injection capillary 601. To the flexible sheet 2. Thereby, the entrainment of gas and the leakage of liquid can be prevented at the time of injection and when the solid injection thin tube 610 is pulled out. However, the pressing pressure of the contact plate 322 is kept within a range in which the solid injection thin tube 610 can move.

Introductory route opening:
As shown in FIGS. 8 (c-1) and (c-2), the pressing screw 331 of the closing plate 321 was loosened by a predetermined amount, and the closing plate 321 was pressed by the outer diameter portion of the injection capillary 601. A part of the flexible sheet 2 is lifted, the flexible sheet 2 is separated from the flow path substrate 1, and the introduction path 314 for injection is opened. However, since the cover plates 3 on both sides of the introduction path opening 313 hold the flexible sheet 2, the introduction path 314 is restricted to a predetermined size. The solid injection thin tube 610 is pushed into the end of the holding channel 111 along the introduction path 314.

Solid injection:
As shown in FIGS. 9D-1 and 9D-2, the piston 612 of the solid injection thin tube 610 is pushed, and the solid specimen 613 is directly transferred from the cylinder 611 into the liquid reagent 614 in the holding channel 111. It is thrown in. At this time, the closed cassette 100 is arranged upright with the insertion guide 121 facing upward, so that bubbles are not generated in the liquid in the holding channel 111.

  The two contact plates 322 are pressed so as to close the gap between the flexible sheet 2 and the injection capillary 601 by tightening the pressing screw 332 by a predetermined amount. It is possible to prevent leakage due to scattering of the liquid reagent 614 when the sample 613 is charged.

Introductory route closure:
As shown in FIGS. 9 (e-1) and 9 (e-2), after the solid specimen 613 is introduced, the solid injection capillary 610 is pulled back to a position before the closing plate 321. Thereafter, the pressing screw 331 of the closing plate 321 is tightened by a predetermined amount. By pressing the closing plate 321, the flexible sheet 2 and the flow path substrate 1 are brought into close contact with each other, the introduction path 314 is closed, and the holding flow path is closed. 111 is sealed.

Pulling out capillary tube for injection:
As shown in FIGS. 7 (f-1) and (f-2), the pulling-out capillary pullout is pulled out in the same manner as the liquid injection step shown in FIGS. 5 (f-1) and (f-2). At this time, when the specific gravity of the solid specimen 613 is larger than that of the liquid reagent 614 filled in advance, the solid specimen 613 is placed under the holding channel 111 as shown in FIG. To settle.

  Although the solid injection step has been described above, injection of a viscoelastic specimen can be performed in the same manner as a solid. In addition, it is possible to inject a liquid by the same method as this solid injection step. In that case, a liquid injection thin tube 601 may be used instead of the solid injection thin tube 610.

<Easy solid injection>
In this way, by separating the flow path substrate 1 and the flexible sheet 2, the solid injection thin tube 610 can be linearly penetrated to the end of the holding flow path 111, and a predetermined amount of reagent is previously obtained. A solid or viscoelastic specimen can be easily injected into the closed cassette 100 being injected.

  Furthermore, the holding channel can be closed by bringing the separated channel substrate 1 and the flexible sheet 2 into close contact with each other.

  In such a solid injection process, in the same manner as the liquid injection process, the pressing operation of the closing plate 321 and the contact plate 322 divided into two parts is used to inject and discharge the liquid and the injection capillary 601. During the drawing operation, bubbles can be prevented from entering the holding channel 111 or liquid can be prevented from leaking.

(Modification 1: Integrated pressing plate)
<Integrated pressure plate>
FIG. 10 is a perspective view separately showing each component of the intermediate block 101 of the closed cassette 100 in the embodiment using the integrated pressing plate 341. For the sake of explanation, only the portion corresponding to the intermediate block 101 is shown.

  The intermediate block 101 shown in FIG. 10 is integrally formed as an entrance / exit pressing means, as shown in FIG. 10, instead of the closing plate 321 and the two-part contact plate 322 shown in FIG. An integral pressing plate 341 is used. The integrated pressing plate 341 is provided separately from the cover plate 3, but is disposed on the same plane as the surface in contact with the flexible sheet 2.

  Hereinafter, the integrated pressing plate 341 will be described with reference to FIG.

  The integrated pressing plate 341 is disposed in the entrance / exit opening 312 and the introduction path opening 313 formed in the cover plate 3. The integrated pressing plate 341 has a close-contact opening 342 at a portion facing the insertion guide 121. The opening of the close contact portion opening 342 of the integrated pressing plate 341 corresponds to a narrow tube close pressing device, and the portion of the integrated pressing plate 341 disposed in the introduction path opening 313 other than the close contact opening 342 is a pressing means for closing the entrance / exit. It corresponds to.

  The integrated pressing plate 341 can be made of a stainless steel plate having a thickness of about 0.1 to 0.3 mm, for example. In this case, it is necessary to sandwich a slide washer 343 made of fluororesin or the like between the pressing screw 344 and the integrated pressing plate 341, so that the integrated pressing plate 341 is under a predetermined pressing by the pressing screw 344. It can be bent by elastic deformation. As a material for the integrated pressing plate 341, it is also possible to use a material that has good sliding properties such as polyacetal and nylon and is easily elastically deformed.

<Injection process>
Each step of the step of injecting a liquid or solid reagent or a sample into the holding channel 111 using the integrated pressing plate 341 and the injection thin tube 601 is shown in FIGS. 11 (a), 11 (b) and 11 (c). The description will be given with reference. 11A, 11B, and 11C are perspective views showing the intermediate block 101 of the closed cassette 100 when the integrated pressing plate 341 is used so that the injection thin tube 601 can be seen through. It is shown.

  Even when the integrated pressing plate 341 is used, if the solid injection capillary 610 is used as the injection capillary 601, a solid or viscoelastic specimen can be injected into the holding channel 111 in the same process.

Inserting capillary tube and sealing around:
As shown in FIG. 11A, the injection capillary 601 is inserted between the flow path substrate 1 and the flexible sheet 2 along the insertion guide 121 and the contact opening 342, and the injection capillary 601 is in close contact. It is made to approach just before the edge part of the part opening 342. At this time, since the integrated pressing plate 341 is tightened by a predetermined amount by the pressing screw 344, the periphery of the injection thin tube 601 is flexible by the integrated pressing plate 341 that forms the outer frame of the close-contact opening 342. It is pressed so as to close the gap between the sheet 2 and the injection capillary 601. Further, even if the outer diameter portion of the injection thin tube 601 is restrained from lifting the flexible sheet 2 under the integrated pressing plate 341, the holding channel 111 is sealed while the introduction channel 314 is closed. Thereby, entrainment of gas and leakage of liquid can be prevented at the time of insertion of the injection thin tube and at the time of pulling out the injection thin tube.

Inlet opening and injection:
As shown in FIG. 11 (b), the injection thin tube 601 enters the holding channel 111 direction beyond the edge portion of the close contact opening 342. At this time, a part of the flexible sheet 2 under the integrated pressing plate 341 is lifted by the outer diameter portion of the injection thin tube 601, and the central portion of the integrated pressing plate 341 is directed outward of the closed cassette 100. It will bend. That is, the integrated pressing plate 341 is not fixed so as to be immovable by the pressing screw 344 shown in FIG. 10, but is fastened via the slide washer 343. Therefore, as shown in FIG. It is deformed by bending outward. When the central portion of the integrated pressing plate 341 is bent outward, the pressure holding the flexible sheet 2 is weakened, and the injection capillary 601 can be further advanced. Therefore, the flexible sheet 2 is separated from the flow path substrate 1, and the introduction path 314 for injection is opened. After the introduction path 314 is opened, a reagent and a specimen are injected into the holding channel 111 from the injection capillary 601 through the introduction path 314. If necessary, the injection capillary 601 or the solid injection capillary 610 further passes through the introduction channel 314 to the end of the holding channel 111 and injects a liquid or an individual specimen into the holding channel 111. can do.

Closing the inlet and pulling the capillary for injection:
As shown in FIG. 11 (c), after injecting the reagent or sample, the injection thin tube 601 is pulled back to a position before the edge portion of the close-contact portion opening 342. At this time, the central portion deflected to the outside of the integrated pressing plate 341 is restored to its original flat shape because the pressure from the inside disappears. Therefore, the flexible sheet 2 and the flow path substrate 1 under the integrated pressing plate 341 are in close contact, the introduction path 314 is closed, and the holding flow path 111 is sealed.

  Next, the injection thin tube 601 is pulled out from between the flow path substrate 1 and the flexible sheet 2. At this time, since the introduction channel 314 is already closed and the holding channel 111 is sealed, bubbles do not enter the holding channel 111 by the drawing operation of the injection thin tube 601.

<Simple operation>
When the central portion of the integrated pressing plate 341 is held so as to be able to bend and deform, the injection is performed without tightening or loosening the screw as in the structure using the closing plate 321 and the two-part contact plate 322. Intrusion of bubbles into the holding channel 111 and leakage of the liquid can be prevented by a simple operation such as simply taking in and out the thin tube 601 for use, and a solid or viscoelastic specimen can easily enter the holding channel 111. Injection can be achieved.

  In the perspective views shown in FIGS. 11A, 11B, and 11C, in order to make it easy to understand the insertion and withdrawal of the injection thin tube 601, it is arranged under the flexible sheet 2 or pressed. It should be noted that the tip of the injection tubule 601 disposed under the plate 341 and the flexible sheet 2 is shown in a transparent manner.

(Modification 2: Elastic body for pressing)
<Elastic body for pressing>
As the entrance / exit path pressing means, a pressing elastic body 351 as shown in FIG. 12 can be used. FIG. 12 is a perspective view showing the components of the intermediate block 101 of the closed cassette 100 using the pressing elastic body 351 separately. For convenience of explanation, only the portion corresponding to the intermediate block 101 is shown in FIG. The pressing elastic body 351 will be described with reference to FIG.

  The pressing elastic body 351 is disposed in the introduction passage opening 313 formed in the cover plate 3, and the close contact portion opening 352 has the same opening width as the introduction passage opening 313. Similar to the flexible sheet 2, the pressing elastic body 351 is made of an elastic material such as a polymer elastomer such as silicon rubber, polypropylene rubber, or urethane rubber. For example, in the case of silicon rubber having a thickness of about 0.3 to 2 mm, the pressing elastic body 351 should have a rubber hardness of JIS-A 40 ° to 60 ° and higher hardness than the flexible sheet 2. Can do. An end of the pressing elastic body 351 is bonded or fastened to the cover plate 3 and is in close contact with the flexible sheet 2. The pressing elastic body 351 may be bonded to the flexible sheet 2 or may be molded integrally with the flexible sheet 2. In the structure shown in FIG. 12, the end of the pressing elastic body 351 is bonded to the cover plate 3.

  The capillary tube pressing means is required to have a function of pressing the flexible sheet 2 against the injection capillary 601 and the flow path substrate 1 so as to close the gap between the periphery of the injection capillary 601 and the flexible sheet 2. . Further, the pressing means for closing the entrance / exit path is required to have a function capable of controlling the deviation and the close contact with the flexible sheet 2 and the flow path substrate 1 that are not fixed as the introduction path 314. Therefore, the flexible sheet 2 is required to be flexible in the vicinity of the insertion guide 121, and needs a certain degree of hardness in the vicinity of the introduction path opening 313. However, since these two requirements cannot be satisfied by one flexible sheet 2, a difference in flexibility and hardness is realized by the pressing means for close contact with the narrow tube and the pressing means for closing the entrance / exit. On the other hand, the flexible sheet 2 is required to be flexible in order for the holding channel 111 to change its volume. Therefore, if the flexible sheet 2 in the vicinity of the introduction path opening 313 has sufficient hardness, it can be a pressing means for closing the access path disclosed in the present invention. Therefore, the pressing elastic body 351 is bonded or closely adhered to the flexible sheet 2 in the introduction path opening 313 or is integrally molded. Further, the contact portion opening 352 has the same opening width as the introduction path opening 313 and has the same function as the opening of the contact portion opening 342 in the integrated pressing plate 341, and the contact portion opening 352 is a pressing means for close contact with narrow tubes. It has become.

<Injection process>
Each step of the step of injecting a liquid or solid reagent or the specimen into the holding channel 111 using the pressing elastic body 351 and the injection thin tube 601 shown in FIG. 12 is shown in FIGS. , (C) will be described. FIG. 13 is a perspective view showing the intermediate block 101 of the closed cassette 100 having a structure using the integrated pressing elastic body 351 so that the injection capillary 601 can be seen through similarly to FIG. .

  The basic operation is the same as that of the intermediate block 101 of the closed cassette 100 having the structure using the integrated pressing plate 341 shown in FIG. 11. If the solid injection capillary 610 is used as the injection capillary 601, A solid or viscoelastic specimen can be injected into the holding channel 111 in the same process.

Inserting capillary tube and sealing around:
As shown in FIG. 13A, the injection thin tube 601 is inserted between the flow path substrate 1 and the flexible sheet 2 along the insertion guide 121 and pressed by the pressing elastic body 351. It is entered just before the site under the flexible sheet 2. At this time, since the pressing elastic body 531 has a predetermined rigidity, the outer diameter portion of the injection thin tube 601 prevents the flexible sheet 2 of the pressing elastic body 531 from being lifted, and the introduction path 314 is closed. The holding channel 111 is sealed as it is. Further, the periphery of the injection capillary 601 is pressed so as to close the gap between the flexible sheet 2 and the injection capillary 601 by the contact opening 352 having the same opening width as the introduction passage opening 313. Accordingly, it is possible to prevent the gas from entering and the liquid from leaking when the injection thin tube is inserted and when the injection thin tube is pulled out.

Inlet opening and injection:
As shown in FIG. 13 (b), the injection thin tube 601 enters the holding flow path 111 in the direction beyond the portion where the pressing elastic body 351 is disposed. At this time, the flexible sheet 2 pressed by the pressing elastic body 351 is lifted by the outer diameter portion of the injection thin tube 601, and at the same time, the pressing elastic body 351 bends outward in the closed cassette 100. When the injection thin tube 601 further enters, the flexible sheet 2 is separated from the flow path substrate 1 and the injection introduction path 314 is opened.

  After the introduction path 314 is opened, a reagent and a sample are injected into the holding channel 111 from the injection thin tube 601 through the introduction path 314. If necessary, the injection capillary 601 or the solid injection capillary 610 passes through the introduction channel 314 and further enters the end of the holding channel 111 to inject a liquid or an individual specimen into the holding channel 111. Also good.

Closing the inlet and pulling the capillary for injection:
As shown in FIG. 13 (c), after injecting the reagent or specimen, the injection capillary 601 is pulled back to a position just before the portion where the pressing elastic body 351 is disposed. At this time, the central portion deflected to the outside of the pressing elastic body 351 is returned to its original state because there is no pressure from the inside. At the same time, the flexible sheet 2 below the pressing elastic body 351 and the flow path substrate 1 are in close contact with each other, the introduction path 314 is closed, and the holding flow path 111 is sealed.

  Next, the injection thin tube 601 is pulled out from between the flow path substrate 1 and the flexible sheet 2. At this time, since the introduction channel 314 is already closed and the holding channel 111 is sealed, bubbles do not enter the holding channel 111 by the drawing operation of the injection capillary 601.

<Simple structure and simple operation>
If the pressing elastic body 351 shown in FIG. 13 is used, it is possible to prevent bubbles from entering the holding flow path 111 and leaking liquid with a simpler structure and a simple operation of simply inserting and removing the injection thin tube 601. Therefore, it is possible to easily inject a solid or viscoelastic specimen into the holding channel 111.

(Second Embodiment)
(Non-penetrating slit)
<Slit shape>
FIG. 14 is a perspective view showing each component of the intermediate block 101 of the closed cassette 100 according to the second embodiment of the present invention separately.

  The intermediate block 101 shown in FIG. 14 has substantially the same structure as that of the intermediate block 101 shown in FIG. 1 except for the shape of the flexible sheet 2. The flexible sheet 2 is provided with a non-penetrating slit 201 at a portion facing the insertion guide 121. In FIG. 14, the non-penetrating slit 201 is illustrated so as to be seen through. When the flexible sheet 2 is made of, for example, silicon rubber, if the thickness of the silicon rubber is about 0.5 mm, the depth of the slit in the thickness direction is about 0.3 mm and is held from the insertion guide 121. The slit length in the direction toward the channel 111 is formed to be about 2 to 5 mm. In addition, the slit 201 is formed in a shape that decreases in depth as it approaches the end point. Since it is desirable that the cut width of the slit 201 be as small as possible, the non-penetrating slit 201 is formed by cutting silicon rubber with a blade, and the slit cut width is zero.

  If the thickness of the silicon rubber is 3 mm or more, it is possible to form a through-hole as used in a well-known self-sealing port. However, the holding channel 111 is fully closed in the flexible sheet 2. It is necessary to satisfy the following two conditions: it must be elastically deformed until it is heated, and it must be heated and cooled via the flexible sheet 2 for the reaction. Silicon rubber with a thickness of 1 mm or more It is not appropriate to use

<Injection process>
Each step of the process of injecting a liquid such as a specimen or a reagent into the holding channel 111 using the non-penetrating slit 201 and the injection thin tube 601 will be described with reference to FIG. 15 (a-1), (b-1), (c-1), and FIGS. 16 (d-1) and (e-1) show the intermediate block 101 along the direction in which the injection capillary 601 is inserted. The cross section of is shown. Here, since the injection thin tube 601 is inserted along the centers of the insertion guide 121 and the holding channel 111, these drawings are intermediate blocks along the center of the insertion guide 121 and the holding channel 111. 101 is also a cross-sectional view of FIG. 15 (a-2), (b-2), and FIG. 15 (e-2) show the approximate center of the contact plate 322 perpendicular to the direction in which the injection capillary 601 is inserted as viewed from the insertion guide 121 side. FIG. 15 (c-2) and FIG. 15 (d-2) are cross-sectional views of the intermediate block 101 in the vicinity, and FIG. 15 (c-2) and FIG. A cross-sectional view of the intermediate block 101 near the approximate center of 321, that is, at the introduction passage opening 313 is shown.

  In the injection process, the closing plate 321 and the contact plate 322 divided into two parts are used, which is the same as the process of referring to FIG. 4 according to the first embodiment, but the non-penetrating slit 201 is used. Therefore, the state of the injection capillary 601 is different. In the description of each step of the following injection process, the description of the same part as the process referring to FIG. 4 is omitted.

  If the solid injection capillary 610 is used as the injection capillary 601, a solid or viscoelastic specimen can be injected into the holding channel 111 in the same process.

Fully closed state before insertion of injection tubule:
FIGS. 15A-1 and 15A-2 show a state before the injection capillary 601 is inserted into the non-penetrating slit 201. FIG. The non-penetrating slit 201 is completely closed because it is regulated by the contact plate 322 divided into two on the left and right sides. Further, since the introduction path 314 is also pressed by the closing plate 321, the introduction path 314 is in a closed state, and the holding channel 111 is sealed.

Inserting capillary tube and sealing around:
As shown in FIGS. 15B-1 and 15B-2, the injection thin tube 601 is inserted into the non-penetrating slit 201 and passes completely through the non-penetrating slit 201 to the front of the closing plate 321. Entered. At this time, as shown in FIG. 15 (b-2), the periphery of the injection capillary 601 is almost completely possible as compared with FIG. 4 (b-2) showing the state without the non-penetrating slit 201. The flexible sheet 2 is in close contact. Therefore, the non-penetrating slit 201 can more reliably prevent the intrusion of bubbles and the leakage of the liquid into the holding channel 111 as compared with the case where it is not provided.

  Further, the flexible sheet 2 and the flow path substrate 1 of the introduction path 314 are maintained in a closed state while being in close contact with each other.

Inlet opening and injection:
As shown in FIGS. 15 (c-1) and (c-2), when the pressure of the closing plate 321 is weakened, the flexible sheet 2 is slightly separated from the flow path substrate 1 and the injection introduction path 314 is opened. Is done. After the introduction path 314 is opened, liquid is injected.

Introductory route closure:
As shown in FIGS. 16D-1 and 16D-2, when a predetermined amount of the liquid 602 enters the holding channel 111, the flexible sheet 2 is pressed by the closing plate 321 so that the flexible sheet 2 becomes the channel substrate 1. The introduction channel 314 is closed and the holding channel 111 is sealed.

Pulling out capillary tube for injection:
As shown in FIGS. 13 (e-1) and 13 (e-2), the injection capillary 601 is pulled out from between the flow path substrate 1 and the flexible sheet 2. The portion of the non-penetrating slit 201 is returned to the original state after the pressure from the injection thin tube 601 disappears.

<Improved leakage prevention>
When the non-penetrating slit 201 is used in this way, the periphery of the injection thin tube 601 is almost completely in close contact with the flexible sheet 2, so that at the time of liquid injection / discharge and the operation of pulling out the injection thin tube 601 In addition, it is possible to more reliably prevent bubbles from entering the holding channel 111 and liquid leakage. Therefore, the self-sealing port used when injecting the liquid into the closed cassette 100 becomes unnecessary, and the closed cassette 100 can be downsized.

(Modification: Through-type slit)
<Slit shape>
FIG. 17 is a perspective view separately showing each component of the intermediate block 101 of the closed cassette 100 according to the first modification of the second embodiment of the present invention.

  In the flexible sheet 2, a penetrating slit 202 extending from a portion facing the insertion guide 121 to a single portion of the holding channel 111 is formed. In FIG. 17, the through-type slit 202 is shown so as to be seen through. Other slit structures and manufacturing methods are substantially the same as the non-penetrating slit 201 described with reference to FIG.

<Pressing bridge>
The cover plate 3 is provided with a pressing relief opening 363 in addition to the holding channel opening 311 and the entrance / exit opening 312, and includes a closing bridge 361 serving as a pressing means for closing an entrance / exit and a close bridge 362 serving as a pressing means for close contact with a narrow tube, The three openings are formed separately. For example, when the flexible sheet 2 is made of silicon rubber having a thickness of about 0.5 to 1 mm and a rubber hardness of JIS-A 20 ° to 30 °, the width of the closed bridge 361 and the close bridge 362 is 1 to About 4 mm, the width of the pressure relief opening 363 is about 1 to 4 mm. The outer edge of the contact bridge 362 is located in the vicinity of the portion where the depression of the insertion guide 121 ends.

<Injection process>
Each step of the process of injecting a liquid such as a specimen or a reagent into the holding channel 111 using the through slit 202 and the injection capillary 601 will be described with reference to FIGS.

  18 (a-1), (b-1), (c-1) and FIGS. 19 (d-1) and 19 (e-1) show the intermediate block 101 along the direction in which the injection capillary 601 is inserted. The cross section of is shown. Here, since the injection thin tube 601 is inserted along the centers of the insertion guide 121 and the holding channel 111, these drawings are intermediate blocks along the center of the insertion guide 121 and the holding channel 111. 101 is also a cross-sectional view of FIG. 18 (a-2), (b-2), and FIG. 19 (e-2) show the approximate center of the contact plate 322 perpendicular to the direction in which the injection thin tube 601 is inserted as viewed from the insertion guide 121 side. FIG. 18 (c-2), FIG. 19 (d-2), and FIG. 18 (e-2) show a cross-sectional view of the intermediate block 101 in the vicinity, and the injection capillary 601 viewed from the insertion guide 121 side is inserted. A cross-sectional view of the intermediate block 101 in the vicinity of the approximate center of the closing plate 321 perpendicular to the direction, that is, the introduction passage opening 313 is shown.

  Since the injection process is only an operation of inserting the injection thin tube 601, it is substantially the same as the case of using the pressing elastic body 351 according to the second modification of the first embodiment shown in FIGS. 12 and 13. Since the through-type slit 202 is used, the state of the injection capillary 601 is different. If the solid injection capillary 610 is used as the injection capillary 601, a solid or viscoelastic specimen can be injected into the holding channel 111 in the same process.

Fully closed state before insertion of injection tubule:
FIGS. 18A-1 and 18A-2 show a state before the injection thin tube 601 is inserted into the through-type slit 202. FIG. The through slit 202 is completely closed because it is pressed by the closing bridge 361 and the close contact bridge 362. Further, the introduction path 314 is also closed because the closing bridge 361 is pressed against the flexible sheet 2, and the holding channel 111 is sealed.

Inserting capillary tube and sealing around:
As shown in FIGS. 18 (b-1) and 18 (b-2), the injection thin tube 601 is inserted into the through-type slit 202 and enters the middle of the pressure relief opening 363. The injection thin tube 601 is inserted between the flow path substrate 1 and the flexible sheet 2 by opening a portion of the penetrating slit 202 pressed by the contact bridge 362. A part of the flexible sheet 2 swells into the pressure relief opening 363 and the entrance / exit opening 312 corresponding to the portion that has been pry open. As shown in FIG. 18 (b-2), the periphery of the injection thin tube 601 is almost completely flexible sheet 2 as compared with FIG. 4 (b-2) showing the state without the through-type slit 202. Is in close contact. Accordingly, the through-type slit 202 can more reliably prevent bubbles from entering the holding channel 111 and liquid leakage as compared with a structure without the through-type slit 202. Further, the flexible sheet 2 of the introduction path 314 is maintained pressed against the flow path substrate 1 by the closed bridge 361, and the closed state is maintained.

Inlet opening and injection:
As shown in FIGS. 18 (c-1) and 18 (c-2), the introduction channel 314 is formed in a portion below the flexible sheet 2 where the injection capillary 601 is further advanced and pressed against the closing bridge 361. And the injection capillary 601 is caused to enter the end of the holding channel 111. Thereafter, the liquid is injected into the holding channel 111. FIG. 20 is a perspective view in a state where the liquid is injected into the holding channel 111 so that the injection thin tube 601 can be seen through. The injection thin tube 601 has reached the end of the holding channel 111 by prying through the portion of the through-type slit 202 pressed by the closing bridge 361. A part of the flexible sheet 2 corresponding to the opened portion is bulged into the pressure relief opening 363 and the holding channel opening 311.

  As shown in FIG. 18 (c-2), the periphery of the injection capillary 601 is almost completely in close contact with the flexible sheet 2 even at the site of the closed bridge 361. Therefore, the through-type slit 202 can more reliably prevent the intrusion of bubbles and the leakage of liquid into the holding channel 111 as compared with the case where there is no slit 202.

Introductory road closure:
As shown in FIGS. 19 (d-1) and (d-2), after injecting the reagent or specimen, the injection capillary 601 is pulled back to the middle of the pressure relief opening 363. At this time, the portion of the penetrating slit 202 pressed by the closing bridge 361 is returned to its original state because the pressure from the injection capillary 601 is lost. At the same time, since it is pressed by the closing bridge 361, the flexible sheet 2 comes into close contact with the flow path substrate 1, the introduction path 314 is closed, and the holding flow path 111 is sealed.

Pulling out capillary tube for injection:
As shown in FIGS. 19 (e-1) and (e-2), the injection thin tube 601 is completely pulled out from the through-type slit 202. At this time, the portion of the through-type slit 202 pressed by the close-contact bridge 362 is restored because the pressure from the injection capillary 601 disappears. At the same time, the flexible sheet 2 is pressed against the flow path substrate 1 by the contact bridge 362, so that the flexible sheet 2 is in close contact with the flow path substrate 1.

<Improvement of leakage prevention by simple configuration>
When the through-type slit 202 is used in this way, a part of the cover plate 3 becomes a narrow tube pressing means and an access path closing pressing means, and the structure is simplified. Furthermore, the injection operation of the liquid and the solid or the viscoelastic body can be performed only by inserting and removing the injection thin tube 601. Even during the liquid injection / discharge and the pull-out operation of the injection capillary 601, the periphery of the injection capillary 601 is almost completely in close contact with the flexible sheet 2 over the entire area of the through slit 202. It is possible to further improve the effect of preventing bubbles from entering the holding channel 111 and liquid leakage. Accordingly, the self-sealing port that has been used when injecting liquid into the closed cassette 100 is unnecessary, and the closed cassette 100 can be downsized.

(Third embodiment)
(Flat tube for injection)
<Structure / Production method>
FIG. 17 is a perspective view showing an overview of a flat injection capillary 620 according to the third embodiment of the present invention.

  The flat type injection thin tube 620 is formed in a substantially elliptical shape in which the outer shape of a known plastic disposable pipette tip is flexible, whereas the circular shape is collapsed. As an example, the flat type injection thin tube 620 has an aspect ratio of about 1: 2 to 5, and is formed, for example, in a shape in which a circle having a height of about 0.2 mm and a width of about 0.7 mm is crushed.

  A mold that conforms to the outer shape as shown in FIG. 21 is manufactured, and a plastic material such as polypropylene or polycarbonate is processed by a well-known injection molding method or the like to produce a target flat injection thin tube 620. it can. Alternatively, a plastic-type disposable pipette tip having a known circular shape and flexibility can be crushed from the outside to produce a target flat-type injection capillary 620.

  If the flat injection capillary 620 is a cylinder and includes a piston inside, a flat solid injection capillary that can inject a solid or viscoelastic specimen can be obtained.

<Pressing cantilever>
FIG. 22 is a perspective view separately showing each component of the intermediate block 101 of the closed cassette 100 using the flat injection thin tube 620 according to the third embodiment of the present invention.

  The flexible sheet 2 is not provided with a slit, and is the same as that used in the first embodiment. The cover plate 3 is provided with a pressure relief opening 375 in addition to the holding channel opening 311 and the entrance / exit opening 312. Between the three openings, a closing cantilever 371 that is a pressing means for closing an entrance / exit and a close cantilever 372 that is a pressing means for close contact with a narrow tube are formed. Closed portion slits 373 are provided between the closed cantilevers 371, and close contact portion slits 374 are provided between the close cantilever 372.

  For example, when the flexible sheet 2 is made of silicon rubber having a thickness of about 0.5 to 1 mm and a rubber hardness of JIS-A 20 ° to 30 °, the width of the closed cantilever 371 and the close contact cantilever 372 is about 0.5 to 4 mm. The width of the pressure relief opening 375 is set to about 1 to 4 mm. The outer edge of the contact cantilever 372 is located in the vicinity of the portion where the depression of the insertion guide 121 ends. Further, the width of the closing portion slit 373 is about 0.5 to 0.7 mm, and the width of the close contact portion slit 374 is about 0.7 to 1 mm wider than the closing slit 373.

<Injection process>
With reference to FIG. 23, each step of the process of injecting a liquid such as a specimen or a reagent into the holding channel 111 using the closed cantilever 371, the close contact cantilever 372, and the flat injection thin tube 620 will be described.

  23 (a-1), (b-1), (c-1), and FIGS. 24 (d-1) and (e-1) show the intermediate block 101 along the direction in which the injection capillary 601 is inserted. The cross section of is shown. Here, since the injection thin tube 601 is inserted along the centers of the insertion guide 121 and the holding channel 111, these drawings are intermediate blocks along the center of the insertion guide 121 and the holding channel 111. 101 is also a cross-sectional view of FIG. 23 (a-2), (b-2), and FIG. 5 (f-2) show the approximate center of the contact cantilever 372 orthogonal to the direction in which the injection capillary 601 is inserted as viewed from the insertion guide 121 side. FIG. 24 (c-2) and FIG. 24 (d-2) are cross-sectional views of the intermediate block 101 in the vicinity, and FIG. 24 (c-2) and FIG. 24 (d-2) are closed cantilevers orthogonal to the direction in which the injection capillary 601 is inserted as viewed from the insertion guide 121 side. A cross-sectional view near the approximate center of 371 is shown.

  In the injection process, since only the operation of inserting the flat type injection thin tube 620 is performed, it is substantially the same as the process in the structure shown in FIG. 17 according to the modified example of the second embodiment. Since the slit 202 is not provided and the closed cantilever 371 and the close contact cantilever 372 are used, the state around the flat injection capillary 620 is different.

  If a flat solid injection thin tube having a piston inside is used as the flat injection thin tube 620, a solid or viscoelastic specimen can be injected into the holding channel 111 in the same process.

Fully closed state before insertion of injection tubule:
FIGS. 23A-1 and 23A-2 show a state before the flat injection thin tube 620 is inserted into the insertion guide 121. FIG. The flexible sheet 2 is pressed by the contact cantilever 372 and the flexible sheet 2 is in close contact with the flow path substrate 1. Further, since the width of the closing portion slit 373 is substantially equal to the thickness of the flexible sheet 2, the introduction path 314 is also sufficiently pressed by the closing cantilever 371 and is in the closed state, and the holding flow path 111 is completely sealed. Yes.

Inserting capillary tube and sealing around:
As shown in FIGS. 23 (b-1) and (b-2), the flat injection thin tube 620 is inserted into the insertion guide 121 and enters to the middle of the pressure relief opening 375. The flat injection thin tube 620 is inserted between the flexible sheet 2 pressed by the contact cantilever 372 and the flow path substrate 1. A part of the flexible sheet 2 corresponding to the opened portion swells into the contact portion slit 374, the pressure relief opening 375, and the entrance / exit opening 312. As shown in FIG. 23 (b-2), the contact cantilever 372 is bent and deformed to the outside of the closed cassette 100. Further, as shown in FIG. 23 (b-2), the flat injection thin tube 620 is flexible, so that it can be adapted to the flat portion of the flow path substrate 1 by the pressing pressure from the close contact cantilever 372. It has been transformed. Furthermore, since the outer shape of the thin tube is flat around the flat type injection thin tube 620, the contact portion slit 374 has a through-type slit 202 shown in the modification of the second embodiment in FIG. Compared with -2), it is in close contact with the flexible sheet 2 to the same extent. For this reason, the effect of preventing the entry of bubbles into the holding channel 111 and the leakage of the liquid is as high as when the through-type slit 202 is used. In addition, since the outer shape of the thin tube is flat and the close contact portion slit 374 is provided, the flat injection thin tube 620 enters without difficulty.

Inlet opening and injection:
As shown in FIGS. 23 (c-1) and (c-2), the introduction channel 314 is provided in a portion below the flexible sheet 2 where the flat injection capillary 620 is further advanced and pressed by the closing cantilever 371. Is passed to the end of the holding channel 111. Thereafter, the liquid is injected into the holding channel 111. FIG. 25 is a perspective view showing a state in which the liquid is injected into the holding channel 111 so that the flat injection thin tube 620 can be seen through.

  The flat injection thin tube 620 is inserted so as to pry between the flexible sheet 2 pressed by the closed cantilever 371 and the flow path substrate 1. A part of the flexible sheet 2 corresponding to the opened portion swells in the closing portion slit 373, the pressure relief opening 375, and the holding channel opening 311. Since the flat injection thin tube 620 is flexible, the flat injection thin tube 620 is deformed by the pressing pressure from the closed cantilever 371 so as to be adapted to the flat portion of the flow path substrate 1. Further, since the outer shape of the thin tube is flat around the flat type injection thin tube 620, the flexible sheet is substantially the same as that of FIG. 18C-2 due to the closing slit 373. 2 is closely attached. Therefore, the effect of preventing the entry of bubbles into the holding channel 111 and the leakage of the liquid is as high as when the through-type slit 202 is used. Further, since the outer shape of the thin tube is flat and there is the closing portion slit 373, the flat injection thin tube 620 can enter without difficulty.

Introductory road closure:
As shown in FIGS. 23 (d-1) and (d-2), after injecting the reagent or specimen, the flat injection capillary 620 is pulled back to the middle of the opening 375. At this time, since the flexible sheet 2 is pressed by the closed cantilever 371, the flexible sheet 2 comes into close contact with the flow path substrate 1, the introduction path 314 is closed, and the holding flow path 111 is sealed.

Pulling out capillary tube for injection:
As shown in FIGS. 23 (e-1) and (e-2), the flat injection thin tube 620 is completely pulled out from the insertion guide 121. At this time, since the flexible sheet 2 is pressed by the contact cantilever 372, the flexible sheet 2 is in close contact with the flow path substrate 1.

<Improvement of leakage prevention by simple configuration>
Thus, in the structure using the flat injection thin tube 620, if a part of the cover plate 3 is formed on the closed cantilever 371 and the close contact cantilever 372 without providing the through slit 202 in the flexible sheet 2, the thin tube The pressing means for close contact and the pressing means for closing the entrance / exit are formed, and the structure is further simplified. Furthermore, the liquid and solid or viscoelastic material can be easily infused by simply inserting and removing the flat injection thin tube 620. Even at the time of liquid injection / discharge and when the injection capillary 601 is pulled out, the periphery of the injection capillary 601 is almost completely in close contact with the flexible sheet 2. The effect of preventing liquid leakage can be further improved. This eliminates the need for the self-sealing port that has been used to inject liquid into the closed cassette 100 in the past, and facilitates downsizing of the closed cassette 100.

  As described above, the present invention is a closed-type nucleic acid detection cassette used for consistently and automatically processing from input of a sample containing nucleic acid to nucleic acid amplification and other necessary processing and detection of a target nucleic acid. And in the technical field of nucleic acid detection systems using the same.

1 is a perspective view schematically showing an overview of a nucleic acid detection cassette including an intermediate block according to a first embodiment of the present invention. It is a disassembled perspective view which isolate | separates and shows each structure of the nucleic acid detection cassette shown in FIG. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block which concerns on 1st Embodiment of invention shown in FIG.1 and FIG.2. (A-1)-(c-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. (D-1)-(f-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. (A) And (b) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the discharge process of the liquid or gas in the intermediate | middle part block shown in FIG. (A) And (b) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the state at the time of the sample injection in the intermediate | middle part block shown in FIG. (A-1)-(c-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the solid injection | pouring process in the intermediate | middle part block shown in FIG. (D-1)-(f-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the solid injection | pouring process in the intermediate | middle part block shown in FIG. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block which uses the integrated pressing plate which concerns on the modification of 1st Embodiment of this invention. It is a general | schematic perspective view which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block using the integrated pressing plate shown in FIG. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block using the elastic body for a press which concerns on the modification of 1st Embodiment of this invention. It is a general-view perspective view which shows the intermediate part block for demonstrating the liquid injection | pouring process in the intermediate part block using the elastic body for a press shown in FIG. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block which concerns on 2nd Embodiment of this invention. (A-1)-(c-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. (D-1)-(e-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block in the intermediate | middle part block which uses the penetration type slit which concerns on the modification of 1st Embodiment of this invention. (A-1)-(c-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. (D-1)-(e-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. It is a perspective view which shows the general view of the intermediate | middle part block for demonstrating the state at the time of the liquid injection | pouring in the intermediate | middle part block using the penetration type slit shown in FIG. It is a perspective view which shows roughly the outline of the flat type | mold injection | pouring thin tube utilized for the intermediate part block which concerns on 3rd Embodiment of this invention. It is a disassembled perspective view which isolate | separates and shows each structure of the intermediate | middle part block which concerns on 3rd Embodiment of this invention using the flat type | mold injection | pouring thin tube shown in FIG. (A-1)-(c-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. (D-1)-(e-2) is principal part sectional drawing which shows the intermediate | middle part block for demonstrating the liquid injection | pouring process in the intermediate | middle part block shown in FIG. FIG. 23 is a main part sectional view schematically showing an intermediate block for explaining a state of liquid injection in the intermediate block shown in FIG. 22;

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Fixed board | substrate, 2 ... Flexible sheet, 3 ... Cover plate, 4 ... Pressing block 100 ... Nucleic acid detection cassette, 101 ... Intermediate | middle part block, 102, 103 ... End part block, 106 ... Detection part block,
111 ... Holding channel, 117 ... Connection channel, 121 ... Insertion guide, 151 ... Contact part opening,
201 ... Non-through-type slit, 202 ... Through-type slit,
311 ... Holding channel opening, 312 ... Entrance / exit channel opening, 313 ... Introduction channel opening, 314 ... Introduction channel, 317 ... Connection channel opening,
321 ... Closure plate, 322 ... Close contact plate, 331, 332 ... Press screw,
341 ... Integrated pressing plate, 342 ... Close contact opening, 343 ... Slide washer, 344 ... Pressing screw,
351... Elastic body for pressing, 352.
361 ... Closed bridge, 362 ... Close contact bridge, 363 ... Pressing relief opening,
371 ... Closed cantilever, 372 ... Close contact cantilever, 373 ... Closed portion slit, 374 ... Close contact portion slit, 375 ... Pressing relief opening,
500 ... nucleic acid detection chip, 520 ... detection flow path seal, 521 ... detection flow path,
601 ... capillary for injection, 602 ... liquid, 603 ... bubble, 604 ... residual liquid,
610 ... capillary for solid injection, 611 ... cylinder, 612 ... piston, 613 ... solid specimen, 614 ... reagent, 615 ... liquid specimen,
620 ... flat injection tube

Claims (7)

In a closed type nucleic acid detection cassette having a flow channel structure capable of performing a series of steps from amplification of nucleic acid contained in a sample to inspection,
A first block formed between a fixed substrate and a flexible sheet and having a holding channel for holding a reagent for nucleic acid amplification, wherein the fixed substrate and the flexible sheet are in close contact with each other, The holding channel is defined by a close contact region surrounding the holding channel, and an insertion guide for inserting a thin tube for injecting the sample into the holding channel is formed on the fixed substrate toward the holding channel. A close contact area extending along the insertion guide to the holding flow path is formed so as to be peelable, and an entrance / exit path is formed between the fixed substrate and the flexible sheet as the thin tube is inserted into the insertion guide. A first block ;
A retraction channel formed between the fixed substrate and the flexible sheet is provided, and the retraction channel is connected to the holding channel via a channel formed between the fixed substrate and the flexible sheet. A second block, and
A nucleic acid detection chip for nucleic acid detection fixed with a seal provided with a detection channel in the second block;
A contact plate portion provided on the flexible sheet on both sides of the insertion guide, and for tightly attaching the thin tube inserted into the insertion guide to the flexible member;
Provided on the flexible sheet between the insertion guide and the holding flow path, allowing the formation of the access path with insertion of the capillary into the insertion guide, and from the insertion guide of the capillary A closing plate portion that closes the entrance / exit path by bringing the flexible sheet into close contact with the fixed substrate as it is pulled out;
A nucleic acid detection cassette comprising: The nucleic acid detection cassette according to claim 1, wherein the insertion guide, the access path, and the holding flow path are linearly arranged. It said flexible member is a nucleic acid detecting cassette according to claim 1, wherein a slit formed toward the holding channel along the insertion guide. In a closed type nucleic acid detection cassette having a flow channel structure capable of performing a series of steps from amplification of nucleic acid contained in a sample to inspection,
A first block formed between a fixed substrate and a flexible sheet and having a holding channel for holding a reagent for nucleic acid amplification , wherein the fixed substrate and the flexible sheet are in close contact with each other, The holding channel is defined by a close contact region surrounding the holding channel, and an insertion guide for inserting a thin tube for injecting the sample into the holding channel is formed on the fixed substrate toward the holding channel. A close contact area extending along the insertion guide to the holding flow path is formed so as to be peelable, and an entrance / exit path is formed between the fixed substrate and the flexible sheet as the thin tube is inserted into the insertion guide. A first block ;
A retraction channel formed between the fixed substrate and the flexible sheet is provided, and the retraction channel is connected to the holding channel via a channel formed between the fixed substrate and the flexible sheet. A second block, and
A nucleic acid detection chip for nucleic acid detection fixed with a seal provided with a detection channel in the second block;
A contact plate portion provided on the flexible sheet on both sides of the insertion guide, and for tightly attaching the thin tube inserted into the insertion guide to the flexible member;
Provided on the flexible sheet between the insertion guide and the holding flow path, allowing the formation of the access path with insertion of the capillary into the insertion guide, and from the insertion guide of the capillary A closing plate portion that closes the entrance / exit path by bringing the flexible sheet into close contact with the fixed substrate as it is pulled out;
Nucleic acid detection cassette having a are used by being mounted,
A pressing means for pressing or releasing the contact plate portion, allowing the capillary tube to be inserted into the insertion guide by releasing the contact plate portion, and pressing the contact plate portion to move the capillary tube to the flexible member. Pressing means for tightly contacting
A closure pressing means to the closure plate portion in the open state or the closed state, to allow the formation of the and out passage in an open state of the closing plate portion, the flexible closed state of the closing plate portion A closing pressing means that closes the access path by bringing a sheet into close contact with the fixed substrate ;
A cassette injection system comprising: The cassette injection system according to claim 3, wherein the insertion guide, the access path, and the holding flow path are linearly arranged .     6. The cassette injection system according to claim 5, wherein an outer shape of at least a portion of the narrow tube inserted into the access path is flat.     The narrow tube is composed of a needle-like portion inserted into the access path of the first region portion and a base portion from which the needle-like portion extends, and this base portion is brought into contact with the nucleic acid detection cassette. The cassette injection system according to claim 5.

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